Canstatin is a newly discovered basement membrane derived endogenous inhibitor of angiogenesis and tumorigenesis. The anti-tumor activity of canstatin, the non-collagenous domain (NC1) of the alpha 3 chain of type IV collagen, is apparently specific for the inhibition of endothelial cell function yet the mechanism of this activity is not clear. This proposal outlines methods to identify a cell surface binding protein/receptor by screening potential integrin receptors in a competitive cell adhesion assay or alternatively, by direct binding and immunoprecipitation of radiolabeled canstatin. Signaling pathways regulated through this candidate receptor will be characterized biochemically upon canstatin treatment. The minimum region necessary for this activity will be determined using site-directed mutagenesis. In addition, downstream targets will be analyzed using cDNA microarrays to identify changes in gene expression in canstatin treated cells. Comparison of the mechanism of several endogenous inhibitors of angiogenesis is likely to provide information not only necessary for the development of effective antiangiogenic drugs but also useful in thoroughly understanding the innate process of angiogenesis and potential variations that exist. This information also potentially leads to the generation of pro-angiogenic drugs needed in the treatment of many vascular diseases (i.e. restenosis, thrombosis, ischemia).